Compositions and methods for the treatment of cast nephropathy and related conditions

ABSTRACT

Provided are novel peptides of Formula SEQ ID No. 1: 
                         (SEQ ID No. 1)         J 1 CysX 1 X 2 X 3 X 4 X 5 X 6 ProX 7 ThrCysJ 2 J 3 (J 4 ) s (J 5 ) t ;                
pharmaceutically acceptable salts thereof, and pharmaceutical compositions thereof, which are which are effective inhibitors of light chains to uromodulin.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/368,260, filed Dec. 2, 2016, which claims priority to U.S. Provisional Application No. 62/380,068 filed Aug. 26, 2016, the entire contents of each of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 21, 2016, is named 119562-03502_SL.txt and is 20,665 bytes in size.

BACKGROUND

Multiple myeloma is a neoplastic disorder characterized by a clonal proliferation of malignant plasma cells, which produce monoclonal immunoglobulins (i.e., antibodies). Acute kidney injury (AKI) is a well-known complication of multiple myeloma with high tumor burden and high amounts of light chains (which are fragments of immunoglobulins) in the urine. AKI affects up to 50% of patients with multiple myeloma, and has important consequences: it exposes patients to the risk of permanent kidney damage (including end stage kidney failure), is associated with a decrease in overall survival, and impacts the treatment of the underlying myeloma.

In the vast majority of cases, AKI is due to myeloma cast nephropathy that results from the precipitation of complexes (called myeloma casts) formed by light chains and uromodulin (a protein normally present in urine) in the distal part of the nephron. See Leung et al., Adv Chronic Kidney Dis, 21: 36-47. These casts obstruct the nephrons, leading to AKI and induce an inflammatory reaction that leads to fibrosis and irreversible chronic kidney damage. The events leading to cast formation have been dissected. See Huang, et al., J Clin Invest, 92: 2975-83, 1993; Huang, et al., J Clin Invest, 99: 732-6, 1997. In particular, different light chains were found to bind to the same peptidic segment of uromodulin (later identified as D8C) and interacted with uromodulin through their CDJ3 (for complementarity-determining region 3). This finding led to the indentation of a CDJ3 sequence derived peptide that blocked the interaction between monoclonal light chains and uromodulin, and inhibited cast formation in vivo. See e.g., Ying, et al., Am J Pathol, 158: 1859-66, 2012.

Recognizing this result, peptides that inhibit the binding of light chains to uromodulin represent are are an attractive targets for combating myeloma cast nephropathy and related conditions.

SUMMARY OF THE INVENTION

Provided herein are peptides and compositions thereof which are effective binding inhibitors of light chains to uromodulin. (See e.g., Table 3). Such peptides include those of Formula SEQ ID No. 1:

(SEQ ID No. 1) J¹CysX¹X²X³X⁴X⁵X⁶ProX⁷ThrCysJ2J³(J⁴)_(s)(J⁵)_(t); or a pharmaceutically acceptable salt thereof, wherein each of X¹, X², X³, X⁴, X⁵, X⁶, X⁷, J¹, J² J³, J⁴, J⁵, s, and t are as defined and described herein.

Also provided are methods of treating myeloma cast nephropathy in e.g., patient populations with severe renal impairment, comprising administering one or more of the peptides or compositions described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the plasma pharmacokinetics following intravenous bolus injection of a peptide of Formula SEQ ID No. 1 in rat.

FIG. 2 illustrates the plasma pharmacokinetics following 2 hour intravenous infusion of a peptide of Formula SEQ ID No. 1 in rat.

FIG. 3 compares the plasma pharmacokinetics of two peptides of Formula SEQ ID No. 1 after intraperitoneal injection in rat, where a.) shows a plot of peptide vs time at various concentrations; b.) shows a plot of steady state concentration (Css) as a function of. dose; and c.) shows a plot of the Area Under the Cure (AUC) as a function of dose.

FIG. 4 illustrates the urine recovery of a peptide of Formula SEQ ID No. 1.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description of Peptides

In one aspect, provided herein are peptides of Formula SEQ ID No. 1:

(SEQ ID No. 1) J¹CysX¹X²X³X⁴X⁵X⁶ProX⁷ThrCysJ²J³(J⁴)_(s)(J⁵)_(t); or a pharmaceutically acceptable salt thereof, wherein

-   -   J¹ is Aha or Arg;     -   X¹ to X⁷ are each independently a natural or non-natural amino         acid;     -   s, and t are each independently 0 or 1;     -   J² and J³ are each independently Lys or Arg;     -   J⁴, if present is Lys or Arg; and     -   J⁵, if present is Lys or Arg.

2. Peptides and Definitions

“Amino acid” refers to an organic compound containing an amine (—NH₂) and a carboxylic acid (—COOH) functional group, usually along with a side-chain specific to each amino acid. Amino acids can be classified according to the core structural functional groups' locations as alpha- (α-), beta- (β-), gamma- (γ-) or delta- (δ-) amino acids; other categories relate to polarity, pH level, and side-chain group type (aliphatic, acyclic, aromatic, containing hydroxyl or sulfur, etc.). In one embodiment, amino acid includes

“Natural amino acid” is used interchangeably with proteinogenic amino acid and refers to the 20 standard amino acids encoded by the universal genetic code along with selenocysteine and pyrrolysine. The 20 standard amino acids encoded by the universal genetic code include glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartate, glutamate, lysine, arginine, and histidine. Unless otherwise specified, when a natural amino acid is defined without indicating the stereochemistry it will be understood that the defined amino acid is present as the L-enantiomer. Thus, when used as part of a formula, the letter A or Ala means L-alanine.

The full name of a natural amino acid and its corresponding three-letter or one-letter code are used interchangeably. For example, glycine is interchangeable with three letter abbreviation Gly or the one letter abbreviation G; alanine is interchangeable with three letter abbreviation Ala or the one letter abbreviation A, valine is interchangeable with three letter abbreviation Val or the one letter abbreviation V, leucine is interchangeable with three letter abbreviation Leu or the one letter abbreviation L, isoleucine is interchangeable with three letter abbreviation Ile or the one letter abbreviation I, proline is interchangeable with three letter abbreviation Pro or the one letter abbreviation P, phenylalanine is interchangeable with three letter abbreviation Phe or the one letter abbreviation F, tyrosine is interchangeable with three letter abbreviation Tyr or the one letter abbreviation Y, tryptophan is interchangeable with three letter abbreviation Trp or the one letter abbreviation W, serine is interchangeable with three letter abbreviation Ser or the one letter abbreviation S, threonine is interchangeable with three letter abbreviation Thr or the one letter abbreviation T, cysteine is interchangeable with three letter abbreviation Cys or the one letter abbreviation C, methionine is interchangeable with three letter abbreviation Met or the one letter abbreviation M, asparagine is interchangeable with three letter abbreviation Asn or the one letter abbreviation N, glutamine is interchangeable with three letter abbreviation Gln or the one letter abbreviation Q, aspartate is interchangeable with three letter abbreviation Asp or the one letter abbreviation D, glutamate is interchangeable with three letter abbreviation Glu or the one letter abbreviation E, lysine is interchangeable with three letter abbreviation Lys or the one letter abbreviation K, arginine is interchangeable with three letter abbreviation Arg or the one letter abbreviation R, histidine is interchangeable with three letter abbreviation His or the one letter abbreviation H, selenocysteine is interchangeable with three letter abbreviation Sec or the one letter abbreviation U, and pyrrolysine is interchangeable with three letter abbreviation Pyl or the one letter abbreviation O. In one embodiment, the term “natural amino acid” or proteinogenic amino acid refers only to the 20 standard amino acids encoded by the universal genetic code, i.e., G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, and H.

“Non-natural amino acid” refers to a non-proteinogenic amino acid that is not found in proteins (e.g., carnitine, gamma-aminobutyric acid, and D-forms of natural amino acids except glycine) or not produced directly and in isolation by standard cellular machinery (e.g., hydroxyproline and selenomethionine). Other examples include, but are not limited to, β-amino acids (β3 and β2), homo-amino acids, alanine derivatives, alicyclic amino acids, arginine derivatives, asparagine derivatives, aspartic acid derivatives, cysteine derivatives, 2,4-diaminobutyric acid, glycine derivatives, isoleucine derivatives, leucine derivatives, lysine derivatives (such as 6-aminohexanoic acid abbreviated herein as Aha), methionine derivatives, norleucine (nL) and norleucine derivatives, phenylalanine derivatives, phenylglycine derivatives, proline and pyruvic acid derivatives, pyroglutamine derivatives, serine derivatives, threonine derivatives, tryptophan derivatives, norvaline derivatives, 2,3-diaminopropionic acid, ornithine derivatives, valine derivatives, linear core amino acids, and N-methyl amino acids. In one embodiment, the term “non-natural amino acid” refers only to D-forms of the 20 standard amino acids encoded by the universal genetic code. These forms include D-Ala, D-Val, D-Leu, D-Ile, D-Pro, D-Phe, D-Tyr, D-Trp, D-Ser, D-Thr, D-Cys, D-Met, D-Asn, D-Gln, D-Asp, D-Glu, D-Lys, D-Arg, and D-His.

The abbreviation “oxC” means a compound of the structure

The abbreviation “Pen” means a compound of the structure

A “D-amino acid” or D-form of an amino acid means that the indicated amino acid is present as the D-enantiomer. Shorthand notation for the D-enantiomer of an amino acid can be represented by an asterisk (*). For example, I* or I*le, wherein * represents a D-amino acid, refers to the D-enantiomer of Ile (isoleucine).

The peptides described herein may be present in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts are art-recognized and include e.g., relatively non-toxic inorganic and organic acid addition salts, or inorganic or organic base addition salts that are suitable for human consumption. Examples of such salts include, but are not limited to, sodium, potassium, calcium, magnesium, acetate, benzoate, bicarbonate, carbonate, citrate, dihydrochloride, gluconate, glutamate, hydrochloride, and tartrate.

The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier, adjuvant, or vehicle that does not adversely affect the pharmacological activity of the peptide with which it is formulated, and which is also safe for human use. Pharmaceutically acceptable carriers that may be used include, but are not limited to, ion exchangers, alumina, aluminum stearate, magnesium stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, dicalcium phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyvinylpyrrolidone-vinyl acetate, cellulose-based substances (e.g., microcrystalline cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose Phthalate), starch, lactose monohydrate, mannitol, trehalose sodium lauryl sulfate, and crosscarmellose sodium, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, polymethacrylate, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

The terms “subject” and “patient” may be used interchangeably, and mean a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment.

The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

An “effective amount” or “therapeutically effective amount” is a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, for example, an amount which results in the prevention of or a decrease in the symptoms associated with a condition that is being treated, e.g., the conditions described herein.

3. Description of Exemplary Peptides

In a first embodiment, provided herein are peptides of the Formula SEQ ID No. 1:

(SEQ ID No. 1) J¹CysX¹X²X³X⁴X⁵X⁶ProX⁷ThrCysJ²J³(J⁴)_(s)(J⁵)_(t); or a pharmaceutically acceptable salt thereof, wherein the variables are as described above.

In a second embodiment, X⁶ in the peptide of Formula SEQ ID No. 1 is not Glu, Thr, Asn, or Asp; and X⁷ is not Glu or Asp, wherein the remaining variables in SEQ ID No. 1 are as described above.

In a third embodiment, X¹ in the peptide of Formula SEQ ID No. 1 is selected from Arg, Lys, His, Pro, Cys, Thr, Ser, Gln, Glu, Leu, Ile, Met, Ala, Val, Gly, n-Leu, Met, Asp, and Ile, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second embodiment. Alternatively, X¹ in the peptide of Formula SEQ ID No. 1 is selected from Arg, Gln, Glu, Leu, n-Leu, Met, Asp, and Ile, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second embodiment. In another alternative, X¹ in the peptide of Formula SEQ ID No. 1 is selected from Gln, Leu, and n-Leu, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second embodiment.

In a forth embodiment, X² in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment. Alternatively, X² in the peptide of Formula SEQ ID No. 1 is selected from Ser, Thr, Cys, Met, Asn, Lys, Gln, His, Arg, Glu, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment. In another alternative, X² in the peptide of Formula SEQ ID No. 1 is selected from Ser, Gln, His, Arg, Glu, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment. In yet another alternative, X² in the peptide of Formula SEQ ID No. 1 is selected from Gln and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second or third embodiment.

In a fifth embodiment, X³ in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment. Alternatively, X³ in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Trp, Phe, Ser, Thr, Cys, Met, Asn, Ala, Lys, His, Glu, Arg, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment. In another alternative, X³ in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Ser, Ala, Glu, Arg, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment. In yet another alternative, X³ is selected from Tyr, Ser, Arg, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, or forth embodiment.

In a sixth embodiment, X⁴ in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment. Alternatively, X⁴ in the peptide of Formula SEQ ID No. 1 is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Asp, Glu, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment. In another alternative, X⁴ in the peptide of Formula SEQ ID No. 1 is selected from Leu, Asp, Gly, Tyr, Glu, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment. In yet another alternative X⁴ in the peptide of Formula SEQ ID No. 1 is selected from Leu, Asp, Tyr, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, or fifth embodiment.

In a seventh embodiment, X⁵ in the peptide of Formula SEQ ID No. 1 is selected from a natural amino acid, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, or sixth embodiment. Alternatively, X⁵ in the peptide of Formula SEQ ID No. 1 is selected from Ser, Thr, Cys, Met, Asn, Gln, Asp, Glu, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, or sixth embodiment. In another alternative, X⁵ in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Ser, Arg, Glu, and Asp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, or sixth embodiment.

In an eighth embodiment, X⁶ in the peptide of Formula SEQ ID No. 1 is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Gln, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment. Alternatively, X⁶ in the peptide of Formula SEQ ID No. 1 is selected from Ile, Leu, Pro, Val, Ala, Gly, His, Lys, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment. In another alternative, X⁶ in the peptide of Formula SEQ ID No. 1 is selected from Ile, Leu, and Arg, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment. In yet another alternative, X⁶ in the peptide of Formula SEQ ID No. 1 is selected from Ile and Leu, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, or seventh embodiment.

In a ninth embodiment, X⁷ in the peptide of Formula SEQ ID No. 1 is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, and His, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment. Alternatively, X⁷ in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Phe, His, Lys, Arg, and Trp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment. In another alternative, X⁷ in the peptide of Formula SEQ ID No. 1 is selected from Tyr, Arg, and Trp, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment. In yet another alternative, X⁷ in the peptide of Formula SEQ ID No. 1 is Tyr, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment.

In a tenth embodiment, s and t are each 0 in the peptide of Formula SEQ ID No. 1, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment.

In an eleventh embodiment, s and t are each 1 in the peptide of Formula SEQ ID No. 1, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth embodiment.

In a thirteenth embodiment, J¹ in the peptide of Formula SEQ ID No. 1 is Aha, wherein the remaining variables in SEQ ID No. 1 are as described above for SEQ ID No. 1 or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh embodiment.

Specific examples of the disclosed peptides are provided in Table 1 and Table 2 below as well as in the EXEMPLIFICATION sections. Pharmaceutically acceptable salts as well as the neutral forms of these peptides are included in the present disclosure.

TABLE 1 SEQ ID No. Sequence 11 AhaCQQSYSIPWTCKK 12 AhaCQQSYSIPYTCKK 13 AhaCQQYDSLPLTCKK 14 AhaCQQYSYLPITCK*K* 15 AhaCQQYSTAPWTCKK 16 AhaCQQYYSAPPTCKK 17 AhaCQQYKNYPWTCKK 18 AhaCQHYDYLPITCK*K* 20 AhaCQQSYSIPWTCK*K* 21 AhaCQQSYSIPYTCK*K* 22 AhaCQQ*SYSIPWTCK*K* 23 AhaCQQS*YSIPWTCK*K* 24 AhaCQQSY*SIPWTCK*K* 25 AhaCQQ*SYSIPYTCK*K* 26 AhaCQQS*YSIPYTCK*K* 27 AhaCQQSY*SIPYTCK*K* 28 AhaCEQSYSIPYTCKK 29 AhaCQESYSIPYTCKK 30 AhaCQQEYSIPYTCKK 31 AhaCQQSESIPYTCKK 32 AhaCQQSYEIPYTCKK 33 AhaCQQSYSEPYTCKK 34 AhaCQQSYSIEYTCKK 35 AhaCQQSYSIPETCKK 36 AhaCQQSYSIPYECKK 37 AhaCRQSYSIPYTCKK 38 AhaCQRSYSIPYTCKK 39 AhaCQQRYSIPYTCKK 40 AhaCQQSRSIPYTCKK 41 AhaCQQSYRIPYTCKK 42 AhaCQQSYSRPYTCKK 43 AhaCQQSYSIRYTCKK 44 AhaCQQSYSIPRTCKK 45 AhaCQQSYSIPYRCKK 46 AhaCQQSY*SIPYTCK*K*K*K* 50 AhaCQQYSYLPITCK*K* 51 AhaCQQYSYLPITCR*R* 52 AhaCQQSYLPITCK*K* 53 AhaCQQYSLPITCK*K* 54 AhaCQQYDLPITCK*K* 55 AhaCQQSYSIPYTCR*R*

TABLE 2 SEQ ID No. Sequence  2 AhaCAAWDDSLNGPVCKK  3 AhaCnLQALRTPLYTCKK  4 AhaCLSADSSGSYLYVCKK  5 AhaCQVWDNSVGVCKK  6 AhaCQSYDNTLSGSYVCKK  7 AhaCQSYDNTLSGSLCKK  8 AhaCQSYDARNV  9 AhaCQSYDHNNQ 10 AhaCQSYDSTNEGVCKK 19 AhaCQHYGSSALTCKK 47 K*K*CQQSY*SIPYTCK*K* 48 AhaPenQQSY*SIPYTPenK*K* 49 oxCCQQSY*SIPYTCK*K*

4. Uses, Formulation, and Administration

In certain embodiments, the present disclosure provides a method of treating a subject (e.g., a human) suffering from myeloma cast nephropathy, comprising administering to the subject a therapeutically effective amount of a peptide of Formula SEQ ID No. 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the amount of peptide of SEQ ID No. 1 is such that it is an effective binding inhibitor of light chains to uromodulin. In certain embodiments, a provided composition is formulated for administration to a subject in need of such composition.

Pharmaceutically acceptable compositions are included and can be administered to humans and other animals by other methods such as e.g., orally, rectally, parenterally, intracisternally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, as an oral or nasal spray, or the like. In certain embodiments, the disclosed peptides may be administered parenterally (e.g., intravenous).

In certain embodiments, the present disclosure also provides a method of treating a subject (e.g., a human) suffering from myeloma cast nephropathy, comprising administering to the subject a therapeutically effective amount of composition comprising a peptide of Formula SEQ ID No. 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. adjuvant, or vehicle. In certain embodiments, the amount of peptide of SEQ ID No. 1 in a provided composition is such that it is an effective binding inhibitor of light chains to uromodulin.

It will be understood that a specific dosage for any particular subject will depend upon a variety of factors, including age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of a provided peptide in the composition will also depend upon the particular peptide in the composition.

The amount of provided peptides that may be combined with carrier materials to produce a composition in a single dosage form will vary depending upon the patient to be treated and the particular mode of administration.

Exemplification

Standard solid phase techniques were employed to synthesize the disclosed peptides. Fmoc (fluorenylmethyloxycarbonyl) chemistry occurred on PEG-base Rink Amide resin, 0.45 mmol/g loading. Side-chain protection of Fmoc amino acid included t-Bu (t-butyl) on Asp, Ser, Tyr and D-Tyr; Trt (trityl) on Asn and Cys; Boc (tert-butyloxycarbonyl) on Lys; Pbf (2,2,4,6,7-pentamethyldihydrobenzofurane) on Arg, Fmoc on K* (i.e., Fmoc-Lys(Fmoc)-OH was used to coupled to the resin).

General Procedure 1. Fmoc Deprotection

Fmoc deprotection was performed using 20% piperidine in DMF (dimethylformamide) or 20% piperidine/0.1% 6-Cl HOBt (Hydroxybenzotriazole) in DMF, 5 min+20 min for each deprotection.

2. Coupling of Fmoc Amino Acids

Acylations were carried out using 3-fold Fmoc amino acids activated with DIC (3eq.) (N,N′-Diisopropylcarbodiimide) in the presence of 6-Cl-HOBt (3eq.). Each coupling reaction took on average 1-3 h for completion. Kaiser test was used throughout for the in-process control of the coupling completion and the Fmoc deprotection. In the case that the Kaiser test was not satisfactory, re-couplings were done using DIC/6-Cl HOBt or HCTU (2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate)/OxymaPure.

3. Resin Cleavage and Peptide Recovery

Peptidyl resin was extensively washed with DCM (dicloromethane) and methanol to remove the trace of DMF and dried at RT overnight. Peptidyl resin was cleavaged with the mixture of TFA/TIS/EDT/H₂O (trifluoracetic acid/triethylsilane/1,2-ethanedithiol/water) (92.5/2.5/2.5/2.5, v/v/v/v) for 2.5-3 h under nitrogen bubbling. Peptides were collected by precipitation in cold diethyl ether/Hexane (1/1, v/v).

4. Purification of Linear Peptide

Crude liner peptide was purified using Phenomenex Gemini C-18 prep column. 21.5 mm×250 mm. The peptide was then dried by lyophilization..

5. Disulfide Bridge Formation

Purified linear peptide was dissolved in 0.1M ammonium bicarbonate (3-5 mg/ml), stand open to air for 2 days. Frequent HPLC testings were done to monitor the cyclization process. After cyclization, use TFA to adjust to acidic. isolated the product by lyophilization.

6. Cyclic Peptide Purification

Peptides were purified using the following conditions. Column: Phenomenex Gemini, 5u, C18, 110A, 250×21.2 mm Mobile phase A: 0.1% TFA in Acetonitrile/H₂O (50/50). Mobile phase B: 0.1% TFA/H₂O. Gradient: 0-100% A in 120 min Wavelength: 229 nm. Flow rate: 16-20 ml/min.

7. Isolation of Human Uromodulin

Uromodulin was precipitated from fresh urine from healthy donors using 0.58M NaCl. The precipitate was then washed with 0.58M NaCl and resuspended in water. The uromodulin was dialyzed extensively against water to remove salt and other contaminants (MWCo=50 KD). Samples were then stored at −20° C. without lyophilization. Good overall purity was seen by gel electrophoresis.

8. General Binding Assays

Enzyme-linked immunosorbant assays (ELISA) were performed on the disclosed peptides. Plates were coated with the following light chain clones overnight at 4° C.: 0.05 uM BER (subtype lambda, isolated from SP2/0 cells), 0.2 uM ROC (subtype kappa, isolated from CHO cells), and 0.3 uM BEN (subtype lambda, isolated from CHO cells). The plates were washed and treated with bovine serum albumin (BSA) overnight at 4° C. For peptide plates, 10 nM biotin-uromodulin was mixed with peptide overnight at 4° C. Plates were then washed and results were revealed using streptaviding-HRP (horseradish peroxidase) and 3,3′,5,5′-tetramethylbenzidine (TMB). Results are shown in Table 3.

TABLE 3 Binding Inhibition SEQ (IC₅₀ uM) ID No. Sequence BER BEN ROC  2 AhaCAAWDDSLNGPVCKK 500 500 500  3 AhaCnLQALRTPLYTCKK 16.0 12.0 14.9  4 AhaCLSADSSGSYLYVCKK 500 66 500  5 AhaCQVWDNSVGVCKK 500 >120 18.5  6 AhaCQSYDNTLSGSYVCKK >120 19 >120  7 AhaCQSYDNTLSGSLCKK 500 500 500  8 AhaCQSYDARNV 500 58.2 500  9 AhaCQSYDHNNQ 500 26.7 500 10 AhaCQSYDSTNEGVCKK 500 500 500 11 AhaCQQSYSIPWTCKK 34.4 3.2 20.8 12 AhaCQQSYSIPYTCKK 22.9 2.5 27.3 13 AhaCQQYDSLPLTCKK 21.3 3.3 31.9 14 AhaCQQYSYLPITCK*K* 17.3 2.8 20.9 15 AhaCQQYSTAPWTCKK 500 16.7 500 16 AhaCQQYYSAPPTCKK 500 14.3 >120 17 AhaCQQYKNYPWTCKK 500 7.3 >120 18 AhaCQHYDYLPITCK*K* 14.8 0.9 13.7 19 AhaCQHYGSSALTCKK 52 3.3 16.4 20 AhaCQQSYSIPWTCK*K* 60.1 2.6 nt 21 AhaCQQSYSIPYTCK*K* 49.8 nt 75.5 22 AhaCQQ*SYSIPWTCK*K* 96.9 nt nt 23 AhaCQQS*YSIPWTCK*K* 54.1 nt nt 24 AhaCQQSY*SIPWTCK*K* 67.8 nt nt 25 AhaCQQ*SYSIPYTCK*K* 35.9 nt 22.0 26 AhaCQQS*YSIPYTCK*K* 30.1 nt 27.5 27 AhaCQQSY*SIPYTCK*K* 27.1 2.8 26.5 28 AhaCEQSYSIPYTCKK 24.9 nt 25.2 29 AhaCQESYSIPYTCKK 26.3 nt 25.5 30 AhaCQQEYSIPYTCKK 26.5 nt 25.9 31 AhaCQQSESIPYTCKK 25.9 nt 24.1 32 AhaCQQSYEIPYTCKK Nt nt 28 33 AhaCQQSYSEPYTCKK >120 nt >120 34 AhaCQQSYSIEYTCKK >500 nt >120 35 AhaCQQSYSIPETCKK >120 nt >120 36 AhaCQQSYSIPYECKK >120 nt >500 37 AhaCRQSYSIPYTCKK 49.8 nt 27.9 38 AhaCQRSYSIPYTCKK 55 nt 26.4 39 AhaCQQRYSIPYTCKK 20.7 nt 22.1 40 AhaCQQSRSIPYTCKK 23.5 nt 26.2 41 AhaCQQSYRIPYTCKK 20.9 nt 21.6 42 AhaCQQSYSRPYTCKK 27.8 nt 29.5 43 AhaCQQSYSIRYTCKK >500 nt >120 44 AhaCQQSYSIPRTCKK 24.9 nt 25 45 AhaCQQSYSIPYRCKK >500 nt >500 46 AhaCQQSY*SIPYTCK*K*K*K* 23.4 nt 35.0 47 K*K*CQQSY*SIPYTCK*K* 32.3 nt 33.5 48 AhaPenQQSY*SIPYTPenK*K* 120.0 nt 120.0 49 oxCCQQSY*SIPYTCK*K* 43.3 nt 41.2 50 AhaCQQYSYLPITCK*K* 17.3 nt nt 51 AhaCQQYSYLPITCR*R* 13.6 nt nt 52 AhaCQQSYLPITCK*K* 69.0 nt nt 53 AhaCQQYSLPITCK*K* 120.0 nt nt 54 AhaCQQYDLPITCK*K* 120.0 nt nt 56 AhaCnLQALRTPLYTCR*R* 10.6 2.0 18.1 nt = not tested

8. Ex Vivo Conditions

Binding inhibition, stability, and solubility data was obtained for peptides of the Formula SEQ ID No. 1. These results are shown in Table 4. MLC₉₀ represents the lowest IC₅₀ at which uromodulin binding to 90% of the light chains tested was inhibited. KBB is kidney brush border enzyme.

TABLE 4 SEQ Binding ID Inhibition Stability Solubility No. MLC₉₀ Plasma_(50%) KBB_(50%) Urine_(80%) (saline, mg/mL) 11 nt  <5 min  5 min >2 hrs ≧100 27 27.1 >120 min >60 min >2 hrs ≧100

9. Pharmacokinetic Evaluations

The plasma pharmacokinetics of SEQ ID No. 27 was evaluated following intravenous (IV) bolus injection (FIG. 1) and 2 hour IV infusion (FIG. 2) in rats. Similarly, FIG. 3 plasma pharmacokinetics of SEQ ID No. 27 following intraperitoneal injection of SEQ ID No. 27 when compared with SEQ ID No. 4 in rat. This data establishes that peptides having SEQ ID No. 1, in particular SEQ ID No. 27, has high efficacy and very good dose-exposure proportionally over a broad range of doses.

Surprisingly, and perhaps most notably, the clearance rate of SEQ ID No. 27 was essentially equal to the glomerular filtration rate, e.g., after infusion of 10 mg/kg of SEQ ID No. 27 for 2 hours to 6 rats, the percentage of intact peptide recovered in the urine was 100%±22% (mean±SD). See FIG. 4.

It will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example. The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art. 

1. A peptide of the Formula SEQ ID No. 1: (SEQ ID No. 1) J¹CysX¹X²X³X⁴X⁵X⁶ProX⁷ThrCysJ²J³(J⁴)_(s)(J⁵)_(t);

or a pharmaceutically acceptable salt thereof, wherein J¹ is Aha or Arg; X¹ to X⁷ are each independently a natural or non-natural amino acid; s, and t are each independently 0 or 1; J² and J³ are each independently Lys or Arg; J⁴, if present is Lys or Arg; and J⁵, if present is Lys or Arg.
 2. The peptide of claim 1, wherein X¹ is selected from Arg, Lys, His, Pro, Cys, Thr, Ser, Gln, Glu, Leu, Ile, Met, Ala, Val, Gly, n-Leu, Met, Asp, and Ile.
 3. The peptide of claim 1, wherein X¹ is selected from Arg, Gln, Glu, Leu, n-Leu, Met, Asp, and Ile.
 4. The peptide of claim 1, wherein X¹ is selected from Gln, Leu, and n-Leu.
 5. The peptide of claim 1, wherein X² is selected from a natural amino acid.
 6. The peptide of claim 1, wherein X² is selected from Ser, Thr, Cys, Met, Asn, Lys, Gln, His, Arg, Glu, and Asp.
 7. The peptide of claim 1, wherein X² is selected from Ser, Gln, His, Arg, Glu, and Asp.
 8. The peptide of claim 1, wherein X² is selected from Gln and His.
 9. The peptide of claim 1, wherein X³ is selected from a natural amino acid.
 10. The peptide of claim 1, wherein X³ is selected from Tyr, Trp, Phe, Ser, Thr, Cys, Met, Asn, Ala, Lys, His, Glu, Arg, and Asp.
 11. The peptide of claim 1, wherein X³ is selected from Tyr, Ser, Ala, Glu, Arg, and Asp.
 12. The peptide of claim 1, wherein X³ is selected from Tyr, Ser, Arg, and Asp.
 13. The peptide of claim 1, wherein X⁴ is selected from a natural amino acid.
 14. The peptide of claim 1, wherein X⁴ is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Asp, Glu, Lys, Arg, and His.
 15. The peptide of claim 1, wherein X⁴ is selected from Leu, Asp, Gly, Tyr, Glu, and Arg.
 16. The peptide of claim 1, wherein X⁴ is selected from Leu, Asp, Tyr, and Arg.
 17. The peptide of claim 1, wherein X⁵ is selected from a natural amino acid.
 18. The peptide of claim 1, wherein X⁵ is selected from Ser, Thr, Cys, Met, Asn, Gln, Asp, Glu, Lys, Arg, and His.
 19. The peptide of claim 1, wherein X⁵ is selected from Tyr, Ser, Arg, Glu, and Asp.
 20. The peptide of claim 1, wherein X⁶ is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Cys, Met, Gln, Lys, Arg, and His.
 21. The peptide of claim 1, wherein X⁶ is selected from Ile, Leu, Pro, Val, Ala, Gly, His, Lys, and Arg.
 22. The peptide of claim 1, wherein X⁶ is selected from Ile, Leu, and Arg.
 23. The peptide of claim 1, wherein X⁶ is selected from Ile and Leu.
 24. The peptide of claim 1, wherein X⁷ is selected from Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Cys, Met, Asn, Gln, Lys, Arg, and His.
 25. The peptide of claim 1, wherein X⁷ is selected from Tyr, Phe, His, Lys, Arg, and Trp.
 26. The peptide of claim 1, wherein X⁷ is selected from Tyr, Arg, and Trp.
 27. The peptide of claim 1, wherein X⁷ is Tyr.
 28. The peptide of claim 1, wherein s and t are each
 0. 29. The peptide of claim 1, wherein s and t are each
 1. 30. The peptide of claim 1, where J¹ is Aha.
 31. The peptide of claim 1, wherein the peptide is selected from AhaCQQSYSIPWTCKK; AhaCQQSYSIPYTCKK; AhaCQQYDSLPLTCKK; AhaCQQYSYLPITCK*K*; AhaCQQYSTAPWTCKK; AhaCQQYYSAPPTCKK; AhaCQQYKNYPWTCKK; AhaCQHYDYLPITCK*K*; AhaCQQSYSIPWTCK*K*; AhaCQQSYSIPYTCK*K*; AhaCQQ*SYSIPWTCK*K*; AhaCQQS*YSIPWTCK*K*; AhaCQQSY*SIPWTCK*K*; AhaCQQ*SYSIPYTCK*K*; AhaCQQS*YSIPYTCK*K*; AhaCEQSYSIPYTCKK; AhaCQESYSIPYTCKK; AhaCQQEYSIPYTCKK; AhaCQQSESIPYTCKK; AhaCQQSYEIPYTCKK; AhaCQQSYSEPYTCKK; AhaCQQSYSIEYTCKK; AhaCQQSYSIPETCKK; AhaCQQSYSIPYECKK; AhaCRQSYSIPYTCKK; AhaCQRSYSIPYTCKK; AhaCQQRYSIPYTCKK; AhaCQQSRSIPYTCKK; AhaCQQSYRIPYTCKK; AhaCQQSYSRPYTCKK; AhaCQQSYSIRYTCKK; AhaCQQSYSIPRTCKK; AhaCQQSYSIPYRCKK; AhaCQQSY*SIPYTCK*K*K*K*; AhaCQQYSYLPITCK*K*; AhaCQQYSYLPITCR*R*; AhaCQQSYLPITCK*K*; AhaCQQYSLPITCK*K*; AhaCQQYDLPITCK*K*; and AhaCQQSYSIPYTCR*R*;

or a pharmaceutically acceptable salt thereof.
 32. A pharmaceutical composition comprising a peptide of claim 1, or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.
 33. A method of treating cast neuropathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a peptide of claim 1, or a pharmaceutically acceptable salt thereof. 